The disaccharide lactose has no specific diffusion pathway across the outer membrane of Escherichia coli. At least three classes of spontaneous mutation affecting outer membrane permeability arose with each of three independent E. coli populations adapting to prolonged lactose limitation in chemostats. Both structural and regulatory mutations affecting OmpF porin predominated in isolates after 210-280 generations of culture. Six types of ompF mutation were found, including in-frame deletions and substitutions at Arg82 and Asp113, all affecting the channel constriction residues of OmpF. Isolates had increased susceptibility to antibiotics and were affected in the quantity of OmpF, LamB and OmpA proteins. A minimum of three or four mutations was evident in all isolates after 280 generations in a lactose-limited environment, in addition to lac mutations defined in previous studies.